Neovascular diseases of the eye are the leading causes of vision impairment in developed nations. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogensis. Of the VEGF receptors it has long been considered that VEGFR2 is the major effector of ocular neovascularization while the contribution of VEGFR1 has only recently been recognized. Our data support a critical role for VEGFR1 in the regulation of VEGFR-2 induced angiogenesis and show that this is dependent on ?-secretase regulated cleavage and intracellular translocation of VEGFR1 (Cai et al 2002;2006). We can show that ?-secretase is a potent regulator of vascular permeability and angiogenesis both in vitro and in vivo and that this effect is dependent on the targeted translocation of the C-terminal domain of VEGFR1 and its association with [unreadable]-catenin and transcription factor Ets-1 respectively. Based on these observations we put forward the following hypothesis:"VEGFR2 driven neovascularization is negatively regulated by the translocation of the intracellular domain of VEGFR1 and that the mobilization, recruitment and binding of ?-secretase to VEGFR1 is critical to this process. We further postulate that the intracellular domain of VEGFR1 regulates vascular permeability through binding to [unreadable]-catenin. We believe that manipulation of the ?-secretase complex and/or VEGFR1 in vivo will reduce vascular permeability and inhibit aberrant retinal neovascularization." Using cultured retinal and choroidal microvascular cells, and where appropriate COS cells, we will a) determine the molecular mechanisms involved in ?-secretase assembly and activation in the plasma membrane and identify the binding and cleavage sites in VEGFR1, b) characterize how ?-secretase phosphorylates the intracellular domain of VEGFR-1 and how this contributes to VEGFR-1 translocation and signaling, c) use fusion proteins consisting of fluorescently labeled VEGR1 in combination with real time imaging to characterize the translocation and intracellular localization of VEGFR-1 following changes in ?-secretase activity, d) use siRNA studies to identify the steps in ?-secretase formation and activation that regulate permeability and angiogenesis in cultured retinal and choriodal microvascular endothelial cells and e) characterize the role of ?-secretase in regulating the binding of VEGFR1 to junctional proteins. We will substantiate the effects of ?-secretase on in vitro permeability and angiogenesis using three animal models of retinal neovascularization (i.e. oxygen-induced retinopathy model, ADCaS model of retinal neovascularization and the laser-induced choroidal neovascularization model) in conjunction with agents that regulate specific ?-secretase subunits to evaluate their contribution. We believe that characterization of the interaction between the ?-secretase complex and VEGFR1, and the subsequent angiogenic response will identify new strategies for regulating vascular permeability and inhibiting aberrant retinal neovascularization in vivo.